Among the studies recently completed to characterize the role of nitrite as a possible therapeutic option in situations where there is a relative deficiency of nitrite bioactivity are our studies with a mouse model of sickle cell anemia that we have been analyzing for a number of years and our studies in collaboration with the Department of Transfusion Medicine of the NIH Clinical Center on changes in nitrite levels in stored blood. The results with the animal model have been published and will not be summarized further here. In previous studies with human red cells, we found that upon removal of these cells from the body, levels of intracellular nitrite fell rapidly with a half life of less than an hour; we devised a preservation solution using ferricyanide, a thiol reagent and a detergent and could permanetly stabilize these levels. With these methods we found that human red cells normally have a nitrite concentration of about 300 nanomolar, while whole blood levels are about one-half of this, suggesting that most blood nitrite is in erythrocytes. Using these methods we have systematically measured nitrite and nitrate levels in stored whole blood, and red cells both with and without leukoreduction, to see the effects of other components of the blood on nitrite production and or consumption. We find that nitrate levels remain very constant at about 30 micromolar but, to our surprise, we find that the initial rapid fall in nitrite levels tapers and for as long as 42 days significant nitrite levels (about 50 nanomolar) remain in the stored red cells. The levels are comparable in all three methods of storage. We are now conducting studies to establish the mechanism of partial nitrite preservation in stored blood and to see if nitrite supplementation improves the properties of these red cells. In addition, several other studies with long term goals of defining clinical uses of nitrite are being done or being planned at present. We find no evidence of a role of S-nitrosated hemoglobin in the proposed storage lesion affecting red blood cells used for transfusion. In collaboration with the NIH Imaging Center we have been examining the effects of changes in nitric oxide levels on blood flow and function in the brains of rodents. We have worked out conditions so that there is no change in systemic or cerebral blood flow with the administration of a nNOS inhibitor but find that their are significant changes in brain function, which is restored with certain nitric oxide donors, including nitrite ions. We are now testing the pharmacological effects of nitrite on brain function and find that nitrite can restore neurovascular coupling. Further it appears that the high levels of intracellular and extracellular ascorbate in the brain may contribute to the reduction of nitrite to generate NO and we are now testing this hypothesis with rodents that can not synthesize ascorbate themselves and thus we can control ascorbate levels by exogenous administration. We have started a project with NHLBI, NINR, and the DTM of the Clinical Center to study the role of NO depletion in causing painful crises in sickle cell anemia patients. We are measuring levels of hemolysis and evidence of NO destruction by cell-free hemoglobin to see if these parameters correlated with manifestations of the disease. We have recently received an NIH Bench/Bedside grant for this work and have developed two clinical protocols which have been approved by the appropriate IRB to administer pain diaries to sickle cell patients, to measure nitrite, nitrate and exhaled NO levels in these patients as well as gene expression profiles in their leukocytes to see if we can identify markers of pain severity. In our project in collaboration with the American Red Cross to study the role of nitrite ions in the viability and storage of human platelets we have found that there is a small change in nitratelevels during five days of room temperature storage and that nitrite levels decrease but only about to 50% levels and then remain stable; some of this appears to be leached from the plastic of the storage bags. We are studying the significance of this finding and the need for nitrite ions in the retention of platelet viability during storage. We have also completed studies of the protective effects of erythropoietin on cardiac and other cells in culture and have shown that this agent protects against hypoxia and other stresses by increasing NO production (as measured by nitrite), especially in endothelial cells, as well as the erythopoeitin receptor itself. We have begun a new project on the contribution of the gut microbiome to the generation of nitrate and nitrite and, most speculatively, on the formation of ammonia. We are studying several bacteria which are found in the mouth and gut at various oxygen levels to work out their overall metabolism of the nitrogen cycle. These reactions must be considered along with dietary intake and the several NO generation paths we previously have studied to understand overall NO species bioactivity. Lastly, as part of this work we have prepared and published two reviews of the state of nitrate and nitrite in the diet as having potential nutritional benefits in protecting against cardiac and other diseases. Although it is too early to know the long effects of such supplements, there is reason to be optimistic that they may be of benefit and the concerns that limited their use in the past were not significant.